Membrane insertion mechanism of the caveola coat protein Cavin1

Significance Caveolae are cholesterol-enriched membrane invaginations linked to severe muscle and lipid disorders. Their formation is dependent on assembly of the protein Cavin1 at the lipid membrane interface driving membrane curvature. In this work, we dissect the mechanism for how Cavin1 binds and inserts into membranes using a combination of biochemical and biophysical characterization as well as computational modeling. The proposed model for membrane assembly potentiates dynamic switching between shielded and exposed hydrophobic helices used for membrane insertion and clarifies how Cavin1 can drive membrane curvature and the formation of caveolae.


IRRA spectra simulation, band fitting parameters and principal component analysis.
IRRA spectra of the protein containing monolayer recorded on a D2O subphase (Sigma) were simulated in the range of the amid I' vibration according to a three-layer model reported by Kuzmin et al. (1). Simulation and fitting of the multicomponent IRRA bands were performed as described in Schwieger et al. (2). The optical constant of the subphase D2O were taken from Berti et al. (3).
The refractive index of the lipid/polymer film was set to 1.41 and its layer thickness to 2 nm. The band positions of the subcomponents were derived from second derivative spectra and revealed the contribution of three amide I' band components centered at 1662, 1642, and 1626 cm -1 . These components were assigned to one random and two α-helical secondary structure elements, respectively (4). According to literature, the lowest component could also be related to β-sheet amid I' vibrations (5). However, neither the X-ray structure nor CD spectra gave any indication for the presence of β-sheet components in the structure of Cavin1. Rather, we assigned the two lower spectral components to water exposed and water shielded faces of the helices, respectively, as reported for other coiled-coil helical arrangements (6). In analogy, we assumed that the amide bonds in helical structures facing the aqueous subphase are more water accessible and absorb at lower wavenumbers (1626 cm -1 ), whereas the interaction with lipid monolayer reduces hydrogen bonds with hydration water leading to a shift to higher wavenumbers (1642 cm -1 ). The band component assigned to amide I' vibrations of unordered structures (1662 cm -1 ) was assumed to originate from isotropically distributed amid bonds and, therefore, simulated with an order parameter of S = 0 (corresponding to a tilt angle θ of 54° with respect to the interface normal). The polar angle between the helix main axis and the transition dipole moments of the helix amid I' vibrations was set to α = 38° (5). The main axis tilt angle of the two lower band components were set to be identical and varied in a least square Levenberg-Marquardt fit to determine the most probable helix orientation. Further fitting parameters were the full-widths at half-height (fwhh) of the three band components as well as the respective absorption coefficients kmax. The spectra were fitted in the range of 1610-1670 cm -1 and at angles of incidence, φ = 30-70° in increments of 4°, where φ = 49, 52, 55 and 58° were omitted from the fit, because of low reflectivity in the range of the Brewster angle. For better understanding and coherence with the MD simulation results, the determined tilt angles θ, which are defined with respect to the interface normal, are translated into inclination angles γ, which are defined in relation to the plane of the interface (γ = 90°-θ). The confidence interval of the fit minimum was calculated as follows: the goodness of the fit was assessed as the sum of square deviations at each inclination angle γ (SSD). These values were weighted by their minimum with (N-p), were N = 360 is the number of fitted data points and p = 6 is the number of fitted parameters: The X values are F-distributed with 1 degree of freedom. In the given conditions all X ≤ 3.87 (SSD ≤ 1.26·10 -5 ) are within the 95% confidence interval.
A set of IRRA spectra was subjected to principal component analysis (PCA) in order to identify subtle changes in the band shape correlated to hydration differences. The PCA was performed on vector-normalized spectra (to exclude contributions from intensity variations to the principal components) in the range of 1693-1770 cm -1 . All spectra were pretreated as follows: i) a reference spectrum of a bare D2O subphase was subtracted from all spectra, ii) a D2O vapor spectrum was subtracted in a way to best reduce the contribution of vibrational-rotational bands in the spectral region of interest, iii) a polynomial rubber band baseline was subtracted from the spectra (Software OPUS, Bruker, Germany). All analyzed spectra were recorded in s-polarization, at various angles of incidence, φ. Four sets of spectra were analyzed in a common PCA: i) and ii) spectra of a DOPC:DOPE and of a DOPC:DOPE:PI(4,5)P2 monolayer before injection of protein (20 mN m -1 , s-polarization, φ = 40°) and ii) and iv) spectra of the respective monolayers after injection of

Computational simulations programs and scripts
Coarse-grained molecular dynamics simulations of unrestrained protein. For the coarsegrained (CG) simulations, Martini 2.2 force field (7,8) was used and the initial PDB structure ID 4QKV (9) was coarse-grained using the Charmm-GUI platform (10,11). No restraint was added to the Martini coarse-grained MD model of the HR1 coiled coil. A similar Martini HR1 domain was utilized in an earlier study (12) where the 4QKV atomistic structure was coarse-grained by using the Martinize.py script (13). The CG lipid membrane was generated using the insane.py script (11). system. An in-house python code was used to calculate the distance between membrane center and the nearest residue from the membrane center. The number of contacts between the membrane lipid molecules and protein helices was obtained using the 'gmx mindist' tool in Gromacs with a cutoff distance of 0.6 nm for each component. All MD simulation snapshot images were generated using the molecular visualization program VMD (14). Images representing the electrostatic surface potential of the HR1was generated using PyMOL.
To calculate the membrane thickness, we have utilized the approach used by Buchout et al. To calculate the distance between the membrane center and amino acids, we first select the amino acid beads of the protein for which we aim to calculate the beads distance. Then we determine the surrounding lipids molecules of that amino acid bead. The average positions of those surrounding lipid molecules and their thickness or inter leaflet distance was then obtain using the code FATSLiM. These values were then utilized to identify the membrane center position. The distance between the membrane center and amino acid beads were then calculated. At a specific simulation time, the minimum distance, !"# , was calculated as the distance between the membrane center and the center of the nearest bead of any protein residues. This !"# was calculated every 10 ns and the average minimum distance, 〈 !"# 〉, represents the average of all the !"# values.

Hydrogen bonds and protein-membrane interactions in all-atom molecular dynamics
simulations. All-atom simulations were performed in order to investigate the formation of the hydrogen bonds between the Cavin1 HR1 (unrestrained) and the membrane. The simulations were performed using CHARMM36m force field (16) in triplicate. The position of the protein, as well as the number of hydrogen bonds formed between the protein and the membrane (including more specific helices-membrane and lipids-protein bonds) were evaluated. The "gmx hbond" module was used to evaluate the number of hydrogen bonds formed between different groups of molecules.
The all-atom simulations were equilibrated and followed by a production run lasting 300 ns. The latter was performed using 2 fs time step. Verlet cutoff scheme was used with the radii of 1.2 nm for both coulomb and van der Waals interactions. PME type of electrostatic interactions representation was applied. Force-switch modifier was also applied to the van der Waals interactions, with a radius of 1.0 nm. Nose-Hoover thermostat was used with a $ =1.0. Parrinello-Rahman barostat with semi-isotropic coupling was used with the % =5.0, compressibility of 4.5×       The MIP value was determined by extrapolation of the Δπ/π0 plot to the x-axis.